In many biomedical and healthcare applications, the rapid determination of the concentration of an analyte in a biological matrix such as blood, plasma, serum, urine, cerebrospinal fluid (CSF), or tissue extracts, is critically important. Traditionally, such measurements have been made via immunoassay-based techniques such as an enzyme linked immunosorbent assay (ELISA). Such techniques rely on the selective binding of an antibody to a target analyte within the sample. In most assays, a second antibody linked to a colorimetric, fluorometric, or radioactivity-based detection system is used to detect the analyte-antibody complex.
Many fully automated devices capable of running immunoassays on biological fluids have been developed and are commonly used in hospital and research settings. While these systems have the advantages of automation and capacity, they also have significant drawbacks. For example, the specificity and selectivity of each assay is only as good as the antibody being used. Many drugs are metabolized by the cytochrome P450 system in the liver or by other metabolic pathways. Usually the metabolite of a drug isn't active, though in certain cases a pro-drug is administered and it is the metabolite that has pharmaceutical activity. In cases where an antibody has reactivity towards both a drug and it's metabolite it is very challenging to develop an immunoassay. The cross reactivity of the antibody means that an aggregate concentration of drug and metabolite(s) will be quantified while it is only one of these species that has clinical relevance. The azole anti-fungal drugs, many anti-convulsive and anti-epileptic drugs, along with many of the calcineurin inhibitors used in transplant patients fall into this category. For classes of drugs such as benzodiazepenes, it is common for the immunoassay to react to the entire class of drugs rather than individual members of the class.
More recently many of the quantitative assays in clinical and research settings traditionally analyzed by immunosorbent assays are being analyzed by mass spectrometry-based techniques. A very common technique is the use of high-pressure liquid chromatography coupled to triple-quadrupole mass spectrometry (HPLC-MS/MS). Mass-spectrometer (MS) based assays have some significant advantages over immunosorbent assays. For example, MS assays typically have a much higher degree of selectivity for a particular analyte than immunoassay-based techniques. The MS-based assay for the determination of Vitamin D concentrations has the ability to differentiate between hydroxyl and di-hydroxy Vitamin D, an important piece of information in a clinical evaluation, while the immunoassay-based techniques are capable of quantifying total Vitamin D concentrations and cannot differentiate between the various forms.
Some mass-spectrometry based techniques may have some limitations, particularly around sample prep and the requirement to use internal standards and external calibrants for absolute quantitation. Samples that contain high concentrations of ionic strength can cause ion suppression within the source region of the mass spectrometer leading to lower signal and poor instrument response. Often samples are fractionated via chromatography, either prior to MS analysis or during the analysis itself through the use of a coupled technique such as HPLC-MS. When complex samples such as blood are being analyzed, often multiple centrifugation or extraction steps may also be required. The requirement for samples prep and standard curve generation often results in samples for mass spectrometric analysis to be analyzed in batch mode. Researchers tend to prefer workflows where samples are accumulated until a level where the effort to generate standard curves and calibrants is amortized by a sufficient number of samples. A downside of this approach is that the turnaround time is dependent on the rate of sample accumulation—if the sample accumulation rate is slow the time to generate results can be long, especially for the samples received early in each batch. While in some applications, the time-to-results for a mass spectrometric analysis may not be important, there are other applications, such as many clinical or healthcare related applications, where the generation of rapid results is critical.
Both immunoassays and mass spectrometry assays have limitations around time-to-answer. Clinical analyzers running immunoassays are highly automated but commonly require 60 to 90 minutes to provide a result. The lack of automation in most mass spectrometry assays adds the requirement of an experienced operator that makes the time-to-answer much more variable. It is common for many mass spectrometry assays to have very long turnaround times. In cases where sample accumulation is slow, the batch mode process often becomes the biggest limiting factor.